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PubMed1996

Different modes of proton translocation by sensory rhodopsin I.

The membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific black lipid membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.

Bacteriorhodopsins↗

PubMed1996

Use of four-colour flow cytometry to evaluate conjugate formation between human peripheral blood mononuclear cells and tumour target cells.

A four-colour flow cytometry technique is described for the determination of the number and phenotype of conjugate-forming mononuclear effector cells from human blood. The discriminatory power of previously described techniques was improved by labelling the effector cells with antibodies that simultaneously identified natural killer (NK), T, and myeloid cells and by labelling the tumour target cells with fluorescein octadecyl ester (FOE), so that cell conjugates could be differentiated from nonbinding effector cells more effectively than by the use of light scatter. The simultaneous characterisation of the three classes of conjugate-forming cells within a heterogeneous human peripheral blood mononuclear cell (PBMC) population made it possible to investigate conjugate formation by each subpopulation without purification, or semipurification (e.g., by monocyte removal), of the subpopulations. The binding of PBMCs to K562 and RPMI 1788 targets was examined. Binding for all three PBMC subpopulations was optimal at 37 degrees C and was negligible at 0 degree C (except for some T cell binding to RPMI 1788 cells). At 37 degrees C, maximum binding was essentially achieved by 10 min. Sodium azide inhibited the majority of the conjugation by NK and T cells, and that inhibition could be removed by washing the cells prior to conjugation, whereas azide had a negligible effect on the binding by monocytes. It appears that effective conjugation by human peripheral blood NK and T lymphocytes requires the operation of an energy-dependent process, differentiating it from conjugation by monocytes.

Azides↗

PubMed1983

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. IV. Relationship between the number of his- bacteria plated and number of his+ revertants scored in the Salmonella mutagenicity test.

Five laboratories participated in a joint ring study to investigate the role of bacterial cell number in the Salmonella mutagenicity test. A strictly standardized protocol, using sodium azide and TA 1535, was developed and employed to test the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with different dilutions of Salmonella typhimurium TA-100 and TA-1535 cultures. All laboratories detected the mutagenic activity of sodium azide with only a 2-fold variation of test results. For MNNG the interlaboratory variation was approximately 5-fold. Decreasing numbers of test bacteria employed resulted in lower numbers of MNNG-induced revertants in all laboratories. The number of preexisting revertants decreased in direct proportion to the reduced cell content, whereas the number of spontaneous revertants was not as greatly affected. A critical amount of test bacteria was required in order to obtain numbers of induced revertants which were equal to twice the number of spontaneous revertants. Two evaluation parameters which may be employed to describe the mutagenicity of a compound are compared.

Animals↗

PubMed1987

Initial adhesion of murine fibroblasts to collagen and fibronectin occurs by two mechanisms.

The adhesion of Balb/c 3T12 cells to fibronectin (FN) and to denatured (DC) or native (NC) collagen is differentially sensitive to divalent cations and to sodium azide. Short-time adhesion (10 min) to FN requires either Mg2+ or Mn2+, whereas only Mn2+ stimulates attachment to DC and NC. Azide treatment only slightly affects adhesion of cells to FN, but strongly inhibits cell attachment to DC and NC. Attachment to any of these substrata is unaffected by monensin and by treatment of the cells with an intracellular fraction, making unlikely the possibility that molecules released by secretion or cell lysis participate in the adhesive process. Soluble collagen inhibits the adhesion of cells to DC and NC, but does not affect adhesion to FN. Finally, rabbit antiserum against collagen binding proteins inhibits cell attachment to NC and DC; the cells, however, attach normally to FN in presence of this antiserum. Taken together, our results support the view that 3T12 cells attach directly to native or denatured collagens and that FN is not required for this process.

Animals↗

PubMed1988

Spindle microtubule dynamics: modulation by metabolic inhibitors.

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.

Antimetabolites↗

PubMed1996

Peroxynitrite augments fMLP-stimulated chemiluminescence by neutrophils in human whole blood.

The neutrophil respiratory burst was examined by the technique of luminol-dependent chemiluminescence (LDCL) triggered by submaximal concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP) in diluted whole blood. We sought to identify the chemical species responsible for LDCL in whole blood, to examine the role of leukotriene B4 (LTB4) and other arachidonic acid metabolites as mediators of the fMLP signaling pathway, and to investigate the effect of peroxynitrite on this response. Both sodium azide and taurine significantly inhibited LDCL (93% inhibition with 100 microM azide, 52% inhibition with 10 mM taurine). More modest inhibition was seen with superoxide dismutase (SOD), catalase, the nitric oxide synthase inhibitor monomethyl-L-arginine (L-NMMA), and with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (AA-861; no effect), and cytochrome P-450 (SKF 525-A) pathways of arachidonic acid metabolism. The nitric oxide donor SIN-1 (1-100 microM) and peroxynitrite (10-300 microM) also augmented fMLP-induced LDCL. The augmentation seen with peroxynitrite and SIN-1 was attenuated by SOD. Despite the increase in LDCL, peroxynitrite caused a dose-related inhibition of fMLP-stimulated LTB4 release. In summary, our results indicate that (1) LDCL elicited by fMLP in diluted whole blood appears primarily mediated by hypochlorous acid derived from myeloperoxidase; (2) pretreatment with the nitric oxide donor SIN-1 or with peroxynitrite augments LDCL; and (3) LTB4 release does not contribute to fMLP-stimulated LDCL or in the modulation of LDCL by SIN-1 or peroxynitrite.

Antioxidants↗

PubMed1986

Influence of skin source, penetration cell fluid, and partition coefficient on in vitro skin penetration.

Using split-thickness pig skin mounted on in vitro skin penetration-evaporation cells, standard conditions were developed to preserve the viability of the skin as judged by its ability to successfully graft to nude mice. The effects of variations from these conditions on the disposition of radioactivity of radiolabeled compounds were determined. No differences in percutaneous penetration were found for N,N-diethyl-m-toluamide, parathion, and progesterone when Tyrode's solution was used in place of tissue culture media. The percutaneous penetration of benzo(a)pyrene on human and pig skin was unaffected by the presence of sodium azide in the tissue culture media; however, with mouse skin, penetration was lower when sodium azide was present. The disposition of radioactivity following topical application of five radiolabeled compounds was similar on fresh skin compared with skin that had been frozen and exposed to ethylene oxide, although variability of the values was greater with the treated skin. The percutaneous penetration of several compounds was determined on skin with and without the epidermis. The penetration of compounds with a lower log P (log octanol-water partition coefficient) increased to a greater extent (e.g., benzoic acid, log P = 2, sixfold increase) than compounds with a higher log P (e.g., DDT, log P = 5, twofold increase). To further validate the use of pig skin, the percutaneous penetration of 11 compounds on pig skin were correlated (r = 0.79) with the values obtained for human skin under standardized in vitro conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Homogeneous populations of Artemia nauplii and their potential use for in vitro testing in developmental toxicology.

Efforts have begun to establish test subjects other than the intact pregnant mammal to serve as models for rapid teratology screens. Artemia nauplii transcending instar I to later instars were examined to determine their potential for indicating chemicals as potential developmental hazards and thus prioritizing them for more extensive in vivo testing. Several criteria selected for assessing the system's potential for screening were the ability to: collect homogeneous populations of instar I nauplii; characterize intermediate development by technically simple measurements; and demonstrate development-related differentials in naupliar vulnerability. Homogeneous populations of nauplii were harvested from a flow-through hatching and cold storage system. Nauplii accumulated in the system are stored at 4 degrees C in a quiescent state with little physical (body length, body water volume) and biochemical (DNA and protein levels) change and thus are maintained at instar I. Intermediate development of nauplii transcending instar I to IV was characterized after the onset of 25 degrees C incubation by measuring changes in drinking activity, body length, body water volume, and DNA and protein levels. During the first day of incubation, development was greatest between 6 and 24 hours of incubation. Development-related differentials in naupliar vulnerability were shown by comparing median lethal concentrations (LC50) estimated for cadmium sulfate (CdSO4), mercuric chloride (HgCl2), and sodium azide (NaN3) at various times during incubation. With cadmium and mercury, LC50s decreased as nauplii aged and developed; whereas, with azide, LC50s did not vary. Developing nauplii were differentially vulnerable to cadmium and inorganic mercury. Artemia nauplii transcending instar I to IV appear useful for indicating chemicals that preferentially interact with developmental events.

Animals↗

PubMed1995

Centrosome behaviour and orientation of centrioles under the action of energy transfer inhibitors.

2,4-dinitrophenol, dinitrophenol together with deoxyglucose, sodium azide and ouabain didn't alter cytoplasmic microtubule (MT) network of cultured PK (pig kidney embryo) cells, meanwhile they induced an increase in the average number of pericentriolar satellites and percentage of centrioles with the primary cilium in these cells. Also all drugs studied increase number of MTs attached to and oriented towards the centrosome. Under the action of ouabain the total number of MTs around the centrosome doubled, meanwhile the number of long MTs emanating from the centrosome increased more than 15 times. Under the action of all drugs studied, except sodium azide, the number of maternal centrioles oriented perpendicularly to the substrate surface increased significantly from that in control cells.

2,4-Dinitrophenol↗

PubMed1996

Control of spoT-dependent ppGpp synthesis and degradation in Escherichia coli.

Escherichia coli has two ppGpp synthetases, PSI and PSII, encoded by the relA and spoT genes. The spoT gene also encodes a ppGpp hydrolase. During exponential growth and under various starvation conditions, the level of ppGpp depends on the balance of ppGpp synthetic and degradative activities of spoT gene products. To find out how these two activities respond to different physiological conditions and to learn about the signals involved in these responses, rates of ppGpp synthesis and degradation were determined in an E. coli B/r delta relA strain during: (1) multiple amino acid deprivation after a nutritional shift-down from glucose amino acids to glucose minimal medium; (2) carbon source starvation after a "glucose runout"; (3) energy starvation by treatment with sodium azide. To each of these conditions, bacteria responded with a similar gradual accumulation of ppGpp, occurring over a period of 20 to 40 minutes, from the basal level of 4 and 24 pmol/OD460 in glucose amino acids and glucose minimal medium, respectively, to about 100 pmol ppGpp/OD460 unit of culture mass. After multiple amino acid deprivation and during azide treatment, the rate of ppGpp synthesis increased and the rate of ppGpp degradation decreased, but in different proportions by the two kinds of treatment. After glucose runout, both ppGpp synthesis and degradation immediately decreased, but the rate of degradation was reduced more, which caused the accumulation of ppGpp despite its reduced synthesis. ppGpp synthesis required continuous protein synthesis, but ppGpp hydrolysis and its control did not: the rate of ppGpp degradation could be instantly up or down-regulated in response to changes in exogenous amino acid or glucose levels in the absence of protein synthesis. The results suggest that PSII is unstable with an average functional lifetime of 40 seconds or less, and that its activity is generated during or shortly after spoT mRNA translation in response to the availability of amino acids. This regulation is responsible for the growth medium-dependent changes in basal levels of ppGpp. ppGpp hydrolysis is controlled, i.e. inhibited, mainly during conditions of physiological stress, such as multiple amino acid deprivation or energy deprivation. This inhibition can be correlated with an inferred accumulation of uncharged tRNA. Since previous reports have indicated that uncharged tRNA inhibits purified SpoT hydrolase in vitro, it is proposed that ppGpp hydrolase activity is, indeed, controlled by the concentration of uncharged tRNA in the cell. Finally, it was found that neither relC, transcriptional regulation of spoT, nor different translation starts of spoT mRNA are directly involved in the environmental control of SpoT hydrolase and PSII activities.

Amino Acids↗

PubMed1982

Energy-dependent lysosomal wrapping mechanism (LWM) during autophagolysosome formation.

The relation of lysosomes to autophagy, in particular the lysosomal wrapping mechanism (LWM) is investigated ultracytochemically from the viewpoint of demonstrations of energy dependency of the LWM. An induction of the LWM was made in mouse subcutaneous histiocytes by subcutaneous administration of ovalbumin. Prior administration of the inhibitor of glycolysis (2-deoxyglucose) alone and of the oxidative phosphorylation (sodium azide or 2,4-dinitrophenol) alone did not prevent the occurrence of the LWM following ovalbumin administration, but a prior administration of a mixture of 2-deoxyglucose and 2,4-dinitrophenol or 2-deoxyglucose and sodium azide prevented the occurrence of the LWM. These results suggest that in order for the LWM to take place ATP is required as an energy source.

2,4-Dinitrophenol↗

PubMed1989

Interaction of mammalian cells with polymorphonuclear leukocytes: relative sensitivity to monolayer disruption and killing.

Monolayers of murine fibrosarcoma cells that had been treated either with histone-opsonized streptococci, histone-opsonized Candida globerata, or lipoteichoic acid-anti-lipoteichoic acid complexes underwent disruption when incubated with human polymorphonuclear leukocytes (PMNs). Although the architecture of the monolayers was destroyed, the target cells were not killed. The destruction of the monolayers was totally inhibited by proteinase inhibitors, suggesting that the detachment of the cells from the monolayers and aggregation in suspension were induced by proteinases releases from the activated PMNs. Monolayers of normal endothelial cells and fibroblasts were much resistant to the monolayer-disrupting effects of the PMNs than were the fibrosarcoma cells. Although the fibrosarcoma cells were resistant to killing by PMNs, killing was promoted by the addition of sodium azide (a catalase inhibitor). This suggests that the failure of the PMNs to kill the target cells was due to catalase inhibition of the hydrogen peroxide produced by the activated PMNs. Target cell killing that occurred in the presence of sodium azide was reduced by the addition of a "cocktail" containing methionine, histidine, and deferoxamine mesylate, suggesting that hydroxyl radicals but not myeloperoxidase-catalyzed products were responsible for cell killing. The relative ease with which the murine fibrosarcoma cells can be released from their substratum by the action of PMNs, coupled with their insensitivity to PMN-mediated killing, may explain why the presence of large numbers of PMNs at the site of tumors produced in experimental animals by the fibrosarcoma cells is associated with an unfavorable outcome.

Animals↗

PubMed1993

In vitro oxidative inactivation of glutathione S-transferase from a freeze tolerant reptile.

We have previously reported that when garter snakes. Thamnophis sirtalis parietalis, a freeze tolerant species, were exposed to 5 h freezing at -2.5 degrees C organs showed increases in the activities of anti-oxidant enzymes, especially catalase in skeletal muscle. This was interpreted to be an adaptation to deal with the potentially injurious postischemic situation of thawing. The present work analyzes in vitro oxidative inactivation of a possible target of postischemic-induced free radical damage, the secondary anti-oxidant defense glutathione-S transferase, and the protective role of endogenous catalase. Approximately 50% of GST activity from snake muscle homogenates was lost within 2 min after addition of H2O2 plus Fe(II) (0.4-2 mM) in media containing azide whereas addition of iron alone resulted in no damaging effects. The opposing effects of dimethyl sulfoxide and EDTA in modifying this process strongly suggested the involvement of .OH radicals in the GST inactivation. A partial recovery of the activity was promoted by mercaptoethanol, indicating that sulphydryl groups oxidation participate in the mechanism of GST inactivation. Pre-incubation of the reaction media containing H2O2 caused protection of the GST activity only in the absence of azide, indicating that endogenous catalase modulates the extent of oxyradical damage. The protective pre-incubation effect was more efficacious when employing homogenates from lung and liver, organs that have higher catalase activities, as well as homogenates from freezing-exposed muscle (that show an 80% increase in catalase activity, compared with control). The protection against GST inactivation observed in muscle from frozen snakes demonstrates that increased anti-oxidant defenses during freezing exposure can be a key factor in controlling in vitro oxyradical damage. The implications for natural freeze tolerance are discussed.

Adaptation, Physiological↗

PubMed1981

Uptake of free and liposome-entrapped 125I-labelled PVP by rat intestinal sacs in vitro: evidence for endocytosis.

The uptake of free and liposome-entrapped 125I-labelled PVP was measured in and intestinal-sac preparation from adult rats. Uptake of the free macromolecule was directly proportional to the substrate concentration and was reduced by colchicine and sodium azide. Uptake of the liposome-entrapped macromolecule was greater than that of the free macromolecule, was not directly proportional to substrate concentration, showed signs of saturation at high liposome concentrations, and was also reduced by sodium azide and colchicine. These results suggest that gut epithelial cells take up the free macromolecule by fluid-phase endocytosis and the liposome-entrapped macromolecule by adsorptive endocytosis.

Animals↗

PubMed1987

An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers. Application to immunoperoxidase studies on eosinophil-rich tissue preparations.

The inhibitory effect of phenylhydrazine and azide combined with either pre-formed or nascent hydrogen peroxide H2O2 upon endogenous peroxidatic activity, expressed by tissue eosinophils in different disease states, was investigated. It was found that whilst endogenous peroxidatic activity due to eosinophils in a Hodgkin's disease and a histiocytosis X case were adequately inhibited by phenylhydrazine combines with pre-formed or nascent H2O2, the eosinophils in the Onchocerca volvulus nodule were either not at all or only partly inhibited by the two regimens. On the other hand, a combination of azide with nascent H2O2 proved consistently effective against this resistant form of endogenous peroxidatic activity. Using human tonsil sections this protocol was shown to be non-deleterious to T4('CD4'), T6('CD1') and T8('CD8') lymphocyte surface antigens as evidenced by the application of a standard indirect immunoperoxidase technique and the relevant monoclonal antibodies.

Antigens, Surface↗

PubMed1990

Promotion and inhibition of catalytic cooperativity of the Ca2+-dependent ATPase activity of spinach chloroplast coupling factor 1 (CF1).

ATP- and ITP-stimulation of the Ca2+-dependent hydrolysis of low concentrations of [gamma-32P]ATP was used as a direct demonstration of catalytic cooperativity in CF1. CF1 activated by epsilon-subunit removal or dithiothreitol, or by the presence of ethanol in the ATPase assay medium, shows pronounced catalytic cooperativity, with maximal stimulation of [gamma-32P]ATP hydrolysis at about 20 microM CaATP. Catalytic cooperativity is diminished by the presence of the epsilon-subunit or by pretreatment of either untreated or epsilon-depleted CF1 with azide (C1/2=30 microM). Both activated and untreated forms of CF1 also exhibit hydrolysis of CaATP by a high-affinity, low-capacity mode of turnover, which is unaffected by any of the preceding treatments and shows normal Michaelis-Menten behaviour. We propose that this high-affinity mode represents unisite catalysis, and that the endogenous inhibitor, epsilon, and the exogenous inhibitor, azide, both act exclusively on cooperative interactions between the catalytic sites.

Adenosine Triphosphate↗

PubMed1981

Effects of ionophore A23187 on calcium fluxes from cultured adrenal cells.

The effect of the calcium ionophore A23128 on calcium fluxes from Y-1 adrenal cortical cells was investigated. Conditions were chosen which are known to result in an inhibition of steroidogenesis (6 . 10(-6) M ionophore and 3 . 10(-4) M extracellular calcium). Calcium efflux from Y-1 cells exhibited two distinct phases. A fast phase which was insensitive to the mitochondrial poison sodium azide and a slow, azide-sensitive phase. The ionophore brought about a rapid increase in the rate of calcium efflux and an 84% reduction in the size of the calcium pool which was associated with the slow efflux phase as well as a reduction in its rate constant. A decrease in the size of the rapidly exchanging calcium pool was also detected. Ethanol, the solvent which was used for the ionophore, slightly increased the rate constant of the rapidly exchanging pool. Conditions which resulted in diminished steroidogenic capacity also brought about a reduction in the size of an energy dependent, intracellular pool. The data is interpreted as being consistent with a hypothesis that the ionophore-induced inhibition of steroidogenesis may be causatively related to the loss of intracellular calcium or to the mechanism which brings about the loss.

Adrenal Glands↗

PubMed1991

Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide.

Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.

Alcaligenes↗